| Objective To observe the effects of excessive fructose intake during pregnancy on the growth and development of fetal rats and the intestinal microflora of neonatal rats,and to preliminarily explore the possible mechanism.Method1.Animal groups: Sexually mature SD rats were fed adaptively for 1 week,and were caged according to the ratio of male and female 2:1.On the next day,a large number of spermatozoa were observed under vaginal secretions smear as mating success.On day 0of pregnancy,female rats were randomly divided into three groups according to body weight,namely control group(CON)glucose group(GLU)fructose group(FRU),with20 rats in each group.Throughout pregnancy,the mothers were treated with drinking water of different ingredients: distilled water for the control group,20% glucose water solution for the glucose group,and 20% fructose water solution for the fructose group.On the 20 th day of pregnancy,10 pregnant rats in each group were randomly selected and killed.Fetal rat,placenta and blood were collected for the detection of various indicators.The remaining female rats and their newborn offspring were sacrificed within 12-24 h after delivery,and the ileum contents of mother and newborn rats were collected for intestinal microflora detection.2.During the experiment,the health status of rats was observed daily,and the average daily food intake and water intake of pregnant rats in each group were measured and weighed weekly.According to the food intake and water intake data of rats in a week,the energy intake level was calculated and the number of fetal rats in each group was recorded,the crown-rump length were measured and the weight of fetal rats and placenta were weighed.3.Hematoxylin-eosin(HE)staining was used to observe the histopathological changes of rat placenta.4.Enzyme-linked immunosorbent assay(ELISA)was used to determine the serum levels of soluble vascular endothelial growth factor receptor-1(sFlt-1)placental growth factor(PLGF)in pregnant mice and to calculate the ratio.5.Serum levels of Uric acid(UA)Malondialdehyde(MDA)Superoxide dismutase(SOD)Catalase(CAT)and Glutathione peroxidase(GSH-Px)in pregnant rats were determined by ELISA assay.6.The levels of UA,MDA,SOD,CAT and GSH-Px in the placenta of pregnant mice were detected by ELISA assay.7.Real-time quantitative PCR(Real-time PCR)was used to determine the mRNA expression level of Kelch-like epichloropropane Kelch sample related protein-1(Keap1)in placental tissues and Nuclear factor erythroid 2-related factor 2(Nrf2)mRNA expression level.8.Western blot(WB)was used to determine the expression level of Keap1 total protein Nrf2 in the placenta of pregnant rats.9.The results of 16S rDNA sequencing showed that the structure of intestinal microflora in the fructose group was changed compared with the control group.There was no significant change in the relative abundance of each Phylum among female rats in each group.The ratio of Firmicutes relative abundance to Bacteroidetes relative abundance of neonatal rats in fructose group was significantly lower than that in control group.Result1.There were no significant differences in energy intake and body weight among all groups during the whole pregnancy(P>0.05).2.There was no significant difference in the number of fetal mice among the three groups.the fetal crown-rump length,fetal weight,placental weight and the ratio of fetal weight to placental weight in fructose group were significantly lower than those in control group and glucose group(P<0.05).3.HE results showed that the placental cells of pregnant rats in the fructose group were disordered and a large number of red blood cells were visible.4.Compared with control group and glucose group,serum sFlt-1 level of pregnant rats in fructose group increased 14.84% and 20.30% significantly(P<0.05),PLGF level was significantly decreased 20.14% and 13.74%(P<0.05),sFlt-1 /PLGF was significantly increased 42.93% and 32.41%(P<0.05).5.ELISA results showed that compared with the control group and glucose group,the serum UA,MDA level in fructose group was significantly increased,while the levels of SOD,CAT and GSH-Px were significantly decreased(P<0.05).6.Compared with the control group and the glucose group,the UA level in the placenta of pregnant rats in the fructose group was significantly increased,but there was no significant difference between the control group and the glucose group(P<0.05).7.PCR results showed that compared with the control group and glucose group,the expression level of Keap1 mRNA was significantly increased by 88.89% and 31.69% in the fructose group,while the expression level of Nrf2 mRNA was significantly decreased by 46.94% and 29.73% in the pregnant mice placenta(P<0.05).8.Compared with the control group,the expression level of Keap1 protein in the placental tissues of pregnant mice in the fructose and glucose groups was significantly increased by 66.34% and 19.15%,while the expression level of Nrf2 nuclear protein was significantly decreased by 39.00% and 20.78%(P<0.05).9.The results of 16S rDNA sequencing showed that the structure of intestinal flora of female rats in the fructose group was changed compared with the control group,and the diversity of intestinal flora was significantly decreased compared with the glucose group(P<0.05);there was no significant difference in the relative species abundance at phylum level and genus level among the three groups(P>0.05).However,compared with the control group,the relative abundance of Firmicutes,Bacteroidetes,Firmicutes/Bacteroidetes and Blautia in female rats in fructose group showed a decreasing trend,the relative abundance of Akkermansia and Proteobacteria showed an increasing trend.The intestinal flora richness of neonatal rats in fructose group was significantly higher than that in control group(P<0.05);compared with the control group,the ratio of the relative abundance of Firmicutes to Bacteroidetes in fructose group was significantly lower than that in the control group(P<0.05);the relative abundance of Firmicutes Bacteroidetes,Bacteroides,Lactobacillus showed a decreasing trend,the relative abundance of Proteobacteria showed an increasing trend,but there was no significant difference(P>0.05).Conclusion1.High fructose exposure during pregnancy may block the expression of antioxidant enzymes by inhibiting Nrf2 nuclear translocation,which puts the placenta in a state of oxidative stress,causes placental dysfunction,and eventually leads to intrauterine growth restriction in fetal mice.2.High fructose exposure during pregnancy can change the structure of intestinal flora in neonatal rats and lead to corresponding changes in the structure of intestinal flora.The adverse change trend of bacterial flora indicates an increased risk of disease. |
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